In Silico Screening Based on Predictive Algorithms as a Design Tool for Exon Skipping Oligonucleotides in Duchenne Muscular Dystrophy

The use of antisense 'splice-switching' oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2'O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R² 0.89) and 53 (R² 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of a target exon

Gradient Injection in Preparative Chromatography: Exploitation of Sample Solvents Different that the Mobile Phase

This work is meant as a contribution to increase the understanding of preparative batch liquid chromatography. It investigates on the use of a different sample solvent for injection that for the elution - the gradient injection. The use of a stronger sample solvent for injection, as often used in pharmaceutical industry, has been given special consideration. For theoretical investigation of the gradient injection, solutions of the equilibrium theory have been applied and extended. This led to identification and derivation of general elution effects of such an injection method. The theoretical results have been verified in two experimental studies. Based on a experimental case study a rather simple and effective methodology has been suggested, suitable for process design and evaluation. It has been shown, that gradient injections have potential for process intensification. Suitable application areas and limits of the gradient injection have been identified

SHADOW® a new welding technique - Basics and applications

The joining processes in watch manufacturing industry are today still dominated by conventional joining techniques like press fitting or crimping. Laser beam micro welding provides consistent joining and high flexibility. Different parts and even different metals can be joined in a non-contact process. The application of a relative movement between the laser beam and the part to be joined at feed rates of up to 60 m/min produces weld seams with a length from 0.6 mm to 15.7 mm using a pulsed Nd:YAG laser with a pulse duration of up to 20 ms. Due to the low energy input, typically 1 J to 6 J, a weld width as small as 50 m and a weld depth as small as 20 m have been attained. This results in low distortion of the joined watch components. Since the first applications of laser beam micro welding of watch components showed promising results, the process could be further enhanced using the SHADOW® welding technique. Aspects of the technique such as tensile strength, geometry a nd precision of the weld been improved. Within the scope of this paper this new welding process will be explained and several examples of joined watch components will be presented with respect to fundamentals and the sustainable implementation of the SHADOW® welding technique into watch manufacturing. New fields of applications for joining different materials such as steel to brass or steel to copper for electrical interconnects will be discussed. Here the SHADOW® welding technique offers new possibilities for the combination of good electrical properties of copper with high mechanical stiffness of steel. The paper will give a closer look to micro joining applications especially using the SHADOW® welding technique. Basics of the process as well as its application on dedicated examples will be shown for small parts such as axis-wheel combinations

Effect on Separation of Injecting Samples in a Solvent Different from the Mobile Phase

The separation of compounds possessing low solubility in the mobile phase could be improved by applying stronger solvents for dissolving the feed. A model system has been investigated with ethanol-water as the mobile phase, Chirobiotic T as the stationary phase and DL-threonine as the sample dissolved in pure water. The adsorption isotherms of DLthreonine were determined as a function of the water content by means of a peak fitting method. Optimal injection conditions of elution chromatography were determined by process simulation. Finally, the optima obtained were verified with experimental data. Copyright © 2013 Elsevier B.V. [accessed 2013 November 26th

Applying Mass Balances to Evaluate the Potential of Hybrid Separation Processes

Within the pharmaceutical industry and in biotechnology there is an increasing need for selective and efficient separation technologies to isolate value added products with a high purity. An important area is for instance the production of enantiomers out of mixtures as they frequently result from synthesis. Often only chromatographic methods yield the desired purity requirements. Unfortunately these methods are rather expensive and their productivity decreased with increasing purity demands1. In contrast, separations based on crystallization are usually less expensive, but often need a certain enrichment to be applicable. The combination of both processes within the design stage gives rise to considerable optimization potential. First general results were already demonstrated, e.g. for the separation of enantiomers2. A possible flowsheet of such a hybrid separation process is shown below. Depending on the concrete separation problem units may be added or subtracted from this scheme. Each of the units involved possess its specific dynamics and limitations. To quantify completely such coupled processes requires large experimental and modelling efforts. Finally it might turn out that the hybrid process (chromatography and crystallization) does not even have the potential to be superior over the single chromatographic separation. In this presentation a general approach based on analytical solutions of mass balance equations will be proposed using several examples for illustration. The advantage of the approach suggested is an a priori evaluation of the potential of a hybrid process for a specific separation problem in the early design stage. The analysis does not need detailed knowledge related to kinetic parameters and other processes involved (e.g. solvent treatment). For the description of coupling chromatography and crystallization no additional tools are required besides a valid model of the chromatographic process and limiting composition necessary for fractional crystallization. With this concept in the early design stage a decision can be drawn whether the coupled process can be more advantageous then chromatography alone. 1. Kaspereit et al., Journal of Chromatography A, 944 (2002), 249-262 2. Lorenz et al., Journal of Chromatography A, 908 (2001), 201-21

Parametric study of coupling chromatography and crystallization for efficient enantioseparations

Within the pharmaceutical industry and in biotechnology there is an increasing need for selective and efficient separation technologies to isolate value added products with a high purity. A hybrid process approach consisting of chromatography and fractional crystallization is studied in detail. The work presented here is concerned with the application and evaluation of the concept of combing these processes to separate a given pharmaceutical intermediate. A comparison performing the same separation exclusively with a single chromatographic process will be given

Applying mass balances to evaluate the potential of hybrid separation processes

Within the pharmaceutical industry and in biotechnology there is an increasing need for selective and efficient separation technologies to isolate value added products with a high purity. An important area is for instance the production of enantiomers out of mixtures as they frequently result from synthesis. Often only chromatographic methods yield the desired purity requirements. Unfortunately these methods are rather expensive and their productivity decreases with increasing purity demands. In contrast, separations based on crystallization are usually less expensive, but often need a certain enrichment to be applicable. The combination of both processes within the design stage gives rise to considerable optimization potential. First general results were already demonstrated, e.g. for the separation of enantiomers. A possible flow sheet of such a hybrid separation process is shown below. Depending on the concrete separation problem units may be added or subtracted from this scheme. Each of the units involved possess its specific dynamics and limitations. To quantify completely such coupled processes requires large experimental and modeling efforts. Finally it might turn out that the hybrid process (chromatography and crystallization) does not even have the potential to be superior over the single chromatographic separation. In this presentation a general approach based on analytical solutions of mass balance equations will be proposed using several examples for illustration. The advantage of the approach suggested is an a priori evaluation of the potential of a hybrid process for a specific separation problem in the early design stage. The analysis does not need detailed knowledge related to kinetic parameters and other processes involved (e.g. solvent treatment). For the description of coupling chromatography and crystallization no additional tools are required besides a valid model of the chromatographic process and the limiting composition necessary for fractional crystallization. With this concept in the early design stage a decision can be drawn whether the coupled process can be more advantageous than chromatography alone

Dynamic analysis of hybrid separation processes: Flowsheet-integration of continuous chromatography and enantioselective crystallisation

Hybrid processes, i.e. processes integrated on the flowsheet level, are a possible method of choice if a certain task is too difficult to be resolved economically using only a single unit operation. Resolving racemates represents a rather difficult separation problem. Although "chiral" chromatography often is the method of choice for enantioseparations, it remains and expensive process. Chiral stationary phases are usually expensive and their performance is limited by their capacity and the separability of the enantiomers. It was shown previously [1,2] that the performance of SMB chromatography improves if the purity requirements can be lowered. This fact can be utilised by coupling the SMB with a second unit operation. In the example studied here, the SMB process supplies a subsequent enantioselective crystallisation with partially resolved mixtures (Fig. 1). Besides the possibility of saving investment costs by using less or lower-priced chiral stationary phase the hybrid process might allow for both higher productivity and lower solvent consumption compared to a stand-alone separation by SMB. The potential arising from the hybrid process can be evaluated on the basis of a few steady state calculations using an SMB model and the mass balances of the hybrid process. This approach is based on the assumption that the SMB process represents the limiting step in terms of separation performance and costs. In comparison to the stand-alone separation by SMB, the integrated process is more complex due to the additional units in the system, their specific dynamic behaviour and the introduction of additional discrete events (e.g., nucleation or seeding). An important issue for a practical realisation of the process is therefore its stability with respect to changes in the operating parameters. To systematically study the system's dynamic behaviour and its stability, a full dynamic mathematical model was developed using the process modelling tool PROMOT [3]. The model equations were solved by the simulation environment DIVA [4]. Essential parts of the model are a continuous chromatography unit modeled as a true-moving bed (TMB) process and the crystallisers assumed to be MDMPR units (mixed suspension - mixed product removal). Population dynamics are modeled using a simple moment formulation. To identify the main characteristics and the parameters with the highest impact on performance and behaviour, steady state continuation calculations and a sensitivity analysis were performed. Parameters revealing a strong impact on both stability and performance are, for example, the design parameters (flow rates) in the continuous chromatography, the reflux ratios of the mother liquor, crystallisation temperatures and enrichment ratios in the solvent treatment. As an example in Fig. 2 is shown how a model process corresponds to a consecutive activation of the recycle streams. The production rates of the crystallisers differ drastically with changes in the recycles. The interplay of nonlinear waves in the chromatographic process results in a complex transient behaviour of all other variables in the system. From the results of the studies above, guidelines for the practical implementation of the flowsheet-integration of SMB and enantioselective crystallisation are derived. [1] H. Lorenz et al., Journal of Chromatography A, 908 (2001), 201-214 [2] M. Kaspereit et al., Journal of Chromatography A, 944 (2002), 249-262 [3] M. Mangold et al., Computers & Chemical Engineering, 28 (2004), 319-332 [4] M. Mangold et al., Chemical Engineering Science, 55 (2000), 441-45

Toxicité intestinale de la fumonisine B1 (conséquences sur la sensibilité des porcelets aux infections)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Using a different solvent for injection than for the mobile phase

In preparative chromatography often the solubility of samples to be separated is low in mobile phases suitable for the separation. Thus, large injection volumes have to be applied, if the same solvent is used for the injection, which leads to reduced resolution. To overcome such limitations in practice, often solvents with higher solubility are used for the injection. However, this introduces the danger of causing undesired effects of peak splitting1,2 and, less frequently, crystallization and blocking phenomena. In this work a methodology is presented to evaluate the use of such an injection strategy. As an example the separation of D and L-threonine on a Chirobiotic-T stationary phase (Astec, U.S.A.) with ethanol/water mixtures as the mobile phases is considered. This methodology consists of: - experimental determination of the adsorption isotherms of the solutes from overloaded chromatograms using a fast peak fitting method3 as a function of the mobile phase composition - determination of the excess isotherm of the components of the mobile phase by perturbations at various mobile phase compositions - determination of a best mobile phase composition by numerical optimization of injections of the solutes: o dissolved in the mobile phase (i.e., cinj=2-16g/l - close to solubility limit) o dissolved in water (cinj=150g/l) An optimal mobile phase composition (25/75 water/ethanol) and optimal injection amounts have been identified. The results of the parametric study have been experimentally verified. For the example studied the high concentration injections in water outperformed by roughly 20% injections in the mobile phase used for the elution. This effect is mainly due to the fact, that much smaller injection volumes can be used to dose the same amount of threonine compared to the injections in the mobile phase. Finally, a discussion on the limits of the application of injections in a different solvent than for the elution will be given. 1P. Janderea, G.Guiochon, J.Chromatogr. 588 (1991) 1-14. 2K. Gedicke, M. Tomusiak, D. Antos, A. Seidel-Morgenstern, J. Chromatogr. A 1092 (2005) 142-148. 3A. Felinger, A. Cavazzini, G. Guiochon, J. Chromatogr. A 986 (2003) 207-225